CN110468095A - A kind of renal epithelial cell and its preparation method and application - Google Patents
A kind of renal epithelial cell and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to stem cell biology fields, and in particular to a kind of renal epithelial cell and its preparation method and application.A kind of renal epithelial cell is disclosed first, expresses CDH1+ and PODXL+, also at least expresses one of CDH2+ and LTL+.The second aspect discloses a kind of method for preparing renal epithelial cell, comprising the following steps: posteriorly intermediate mesoderm differentiation, rear intermediate mesoderm are steeped to kidney duct shape aggregation precursor differentiation, kidney duct shape aggregation precursor to the differentiation of kidney bubble, kidney to renal epithelial cell differentiation multipotential stem cell to the differentiation of kidney progenitor cells, kidney progenitor cells backward for original cell differentiation, the former cell in rear portion.Preparation method differentiation efficiency provided by the invention is high, differentiation effect is stablized, and is suitable for a variety of hPSC cell strains.Breaking up obtained nephrons progenitor cells can satisfy the scientific experiments demands such as kidney diaseases model foundation, the test of renal drug toxicity and drug screening relevant to kidney trouble.
Description
Technical field
The present invention relates to stem cell biology fields, and in particular to a kind of renal epithelial cell and its preparation method and application.
Background technique
Kidney is organ important in human body, has complicated space structure.As the component part of urinary system, kidney
Major function be the ingredient and volume for adjusting body fluid, and remove metabolic waste.Mature kidney can by nephrons to blood into
Row filters and repeatedly recycles the substance needed in human body and finally generate urine and excrete.Furthermore kidney also has adjusting blood
Pressure, maintains the functions such as internal acid-base balance.
Human pluripotent stem cells (hPSC, including human embryo stem cell hESC and people induce multi-potent stem cell hiPSC) is broken up
For kidney progenitor cells and kidney organoid be research human kidney's development, hereditary Kidney Diseases, injury of kidney, renal toxicity test and
The directions such as kidney regeneration provide a new in vitro study platform.The hair of current ureteric bud cell and rear kidney mesenchymal cell
It is gradually clear to educate process, but the growth course of other types the cell such as endothelial cell and interstitial cell in renal tissue is still
It is unclear.By studying vitro differentiation process of the hPSC to kidney organoid, us can be helped further to deepen to internal kidney
The understanding of dirty development.It can study that Congenital Renal is abnormal by the cell of each differential period simultaneously and nephrotoxic drugs
Screening.There are the hereditary Kidney Diseases being clearly mutated existing more than 160 kinds at present.Broken up by the iPSC of heredity nephrotic
Kidney organoid is obtained, kidney diaseases model can be constructed, probes into the pathogenesis of heredity nephrosis.In conjunction with CRISPR/Cas9 base
It, can be in the case where guaranteeing other genetic background unanimous circumstances to saltant type and parent because editing technique introduces targeted mutagenesis to iPSC
The organoid that cell strain differentiates is compared.The gradually loss of nephrons is the morbidity one for leading to chronic renal disease (CKD)
A major reason.The treatment means in the disease advanced stage predominantly dialysis and kidney transplant at present.Only in embryonic development period, kidney just meeting
New nephrons is constantly formed, the nephrons number of kidney is not further added by after newborn's birth.Broken up by the iPSC in patient source and is generated
The free of contamination nephrons progenitor cells of high quality simultaneously perhaps can be used as the tissue that compatibility is immunized by expanded in vitro culture, keep CKD sick
People can generate new nephrons again, to reach the therapeutic effect to CKD.
Have with the immediate article of the present invention: Taguchi etc. reports the rear kidney mesenchyma of 60% or so of relative efficiency
The induced efficiency of cell (SIX2+ cell), these rear kidney mesenchymal cells induced are further divided into nephrons structure.However
They have to rear kidney mesenchymal cell type, without collecting pipe, renal interstitial and endothelial cell types.Takasato etc. is differentiated
Kidney organoid not only include collecting pipe and nephrons also include interstitial cell and endothelial cell network, but intermediate mesoderm to
Kidney organoid breaks up that this process is not fully apparent, and the cost for carrying out induction using the FGF9 of high dose is too excessively high
It is high, and the building of disease model is not also referred to.The differentiation scheme of the propositions such as Morizane successfully obtains 90% or so
SIX2+ cell, and two kinds of training methods of 2D and 3D is further taken to differentiate with each section of renal tubule (including proximal tubule, henry
Li Shi ring, distal tubule) and glomerular podocyte nephrons structure and response can be made to nephrotoxic drugs, cause cell
Apoptosis.For ureteric bud cell, other cell types in the kidneys such as interstitial cell and endothelial cell, the program is not mentioned
And.
Have with the immediate patent of the present invention: Method of differentiation of pluripotent stem
cell for forming kidney organoids.This patent describe a kind of kidney classes that hPSC is divided into vascularization
Organ, the program use Wnt agonist, FGF signal activation agent, heparin, retinoic acid, the induction point of the plurality of reagents such as RA antagonist
Change, cost is excessively high.
Each renal epithelial cell differentiation scheme existing at present is different in process and principle in differentiation scheme, these
Differentiation scheme has the disadvantages that 1) differentiation cost is excessively high, and the differentiation period is long；2) medium component complexity source is indefinite；3)
Differentiation efficiency is low to be unable to get more renal epithelial cells.
Summary of the invention
In order to solve above-mentioned existing technical problem, the present invention provides one kind quickly, efficiently, easily to determine from hPSC
To the novel processing step for being divided into renal epithelial cell.Preparation method differentiation efficiency provided by the invention is high, differentiation effect is stablized,
Differentiation pathway is induced suitable for a variety of hPSC cell strains and 2D or 3D.The nephrons that preparation method is broken up through the invention
Structure and kidney organoid can be used for the foundation of kidney diaseases model, the test of renal drug toxicity and relevant to kidney trouble
Drug screening etc..
Technical solution of the present invention and the prior art have a following difference: first, it adjusts used in each signal path in this programme
Micromolecular inhibitor and cell factor unlike the prior art, ingredient is simple, have more efficient and inexpensive characteristic；
Second, the time used in mature renal epithelial cell is obtained using present invention differentiation and is shortened than the prior art, to a certain degree
On save time cost；Third, this programme are suitable for 2D or 3D culture simultaneously, and method produced according to the present invention is under 2D culture
Break up obtained renal epithelial cell structure, there is the assembling structure similar with internal nephrons structure；4th, the present invention provides
3D culture scheme can be carried out in various specifications culture bottle, can more efficiently obtain greater number of kidney organoid ball
Body, and the maturation time of renal epithelial cell is advanced by 4-5 days.
The present invention has the following advantages: first, preparation method provided by the invention is easy to operate, can lead within the shorter time
3D is crossed to break up to obtain the kidney organoid with mature renal epithelial cell structure；Second, the preparation method provided through the invention
Break up obtained nephrons progenitor cells efficiency and reaches 90% or more；Third, the supplement provided in preparation method of the present invention (add
Add agent) definite ingredients, realize differentiation efficiently, inexpensive；4th, preparation method provided by the invention can be suitable for a variety of
The differentiation of hPSC cell strain.5th, kidney progenitor cells frozen stock solution provided by the invention can effectively freeze differentiation the and obtain for 7-8 days
Kidney progenitor cells.And motility rate is high after recovering, and kidney progenitor cell marker index is still expressed, have continue to be divided into kidney epithelium it is thin
The potential of born of the same parents.
Specifically, technical scheme is as follows:
First aspect of the present invention discloses a kind of renal epithelial cell, expresses CDH1+ and PODXL+, also at least expresses
One of CDH2+ and LTL+.
The second aspect of the present invention discloses a kind of method for preparing renal epithelial cell, comprising the following steps:
S1: multipotential stem cell posteriorly former cell differentiation；
S2: intermediate mesoderm breaks up rear portion original cell backward；
S3: rear intermediate mesoderm breaks up to kidney progenitor cells；
S4: kidney progenitor cells assemble precursor differentiation to kidney duct shape；
S5: kidney duct shape assembles precursor and steeps differentiation to kidney；
S6: kidney is steeped to be broken up to renal epithelial cell.
It preferably, further include S0 before S1: the culture of multipotential stem cell.
Preferably, the S1 includes:
S11: the supernatant of human pluripotent stem cells is absorbed, and renal epithelial cell differentiation complete medium A induction differentiation is added,
The renal epithelial cell differentiation complete medium A includes Wnt signal path activator, BMP4 signal pathway inhibitor and BMP4 letter
Number Pathway Activation agent；
S12: replacing culture medium daily, cultivates 4-5 days.
Preferably, the S2 includes:
S21: absorbing the supernatant of the former cell in rear portion, and renal epithelial cell differentiation complete medium B induction differentiation, institute is added
Stating renal epithelial cell differentiation complete medium B includes TGF-β signal path activator；
S22: replacing culture medium daily, cultivates 2-3 days.
Preferably, the S3 includes:
S31: renal epithelial cell differentiation complete medium C induction differentiation, institute is added in the supernatant of intermediate mesoderm after absorption
Stating renal epithelial cell differentiation complete medium C includes FGF signal path activator；
S32: replacing culture medium daily, cultivates 2-3 days.
It is furthermore preferred that further including step S33: the kidney progenitor cells that differentiation obtains are frozen.
In some currently preferred embodiments of the present invention, kidney progenitor cells are frozen using frozen stock solution in S33, it is described to freeze
Liquid includes the DMSO and recombinant human serum albumin that percentage by volume is respectively 5-20% and 0-10%.
In some embodiments of the invention, the component of above-mentioned frozen stock solution is as shown in table 1 below.
Table 1
Preferably, S4 culture by the way of 2D, comprising the following steps:
S41: absorbing the supernatant of kidney progenitor cells, and renal epithelial cell differentiation complete medium D induction differentiation, the kidney is added
It includes FGF signal path activator and Wnt signal path activator that epithelial cell, which breaks up complete medium D,；
S42: replacing culture medium daily, cultivates 2-3 days.
Preferably, S4 culture by the way of 3D, comprising the following steps:
Kidney progenitor cells are digested, is resuspended with the renal epithelial cell differentiation complete medium D containing Rock inhibitor, is placed in three
Tie up the culture that suspends on shaking table.
Preferably, the S5 includes:
S51: absorbing the supernatant of kidney duct shape aggregation precursor, and renal epithelial cell differentiation complete medium C induction differentiation is added,
The renal epithelial cell differentiation complete medium C includes FGF signal path activator；
S52: replacing culture medium daily, cultivates 2-3 days.
Preferably, the S6 includes:
S61: absorbing the supernatant of kidney bubble, and renal epithelial cell differentiation basal medium E induction differentiation is added；
S62: replacing culture medium daily, cultivates 5-7 days.
It is furthermore preferred that the renal epithelial cell differentiation basal medium E includes glutamine, human transferrin and pancreas islet
Element.
The flow chart of the method for above-mentioned motor neuron is prepared in the present invention as shown in Figure 1, specifically, operation is as follows:
One, human pluripotent stem cells are to renal epithelial cell directed differentiation -2D
(1) Day-3-0: the culture of multipotential stem cell
The undifferentiated multipotential stem cell that the degree of polymerization reaches 70-80% is digested to completely with Trypsin or Accutase
It is unicellular, with certain density be resuspended in appropriate volume multipotential stem cell maintain culture medium in, certain density Rock is added
Inhibitor, and be seeded on Matrigel plate, 37 DEG C are placed in, 5%CO2Concentration, the interior culture of the incubator of saturated humidity.
Day-3-0 optimum experimental:
1. multipotential stem cell used in experiment by stringent versatility verifying (express various versatility markers, and
Can be formed in immune-deficient mice body comprising it is interior, in, the teratoma of outer three germinal layers).Multipotential stem cell maintains training at it
Support and normally cultivated in base, the similar culture medium of culture medium E8 or TeSR or other used, it is optimized after using Nova culture medium into
Row culture can stablize and maintain the long-term cultivation of hPSC in vitro.
2.Rock inhibitor includes Y-27632, Blebbistatin, HA100 etc..Preferably, Rock inhibitor is Y-
27632, concentration is 10 μM；
The inoculum density of 3.hPSC can be 1-10 × 104cells/cm2。
4. cell dissociation buffer includes Trypsin, Accutase, EDTA and other cell dissociation buffers.Preferably, it uses
Accutase carries out cell dissociation.Cell facilitates counting in unicellular after vitellophag, and small to cell damage.
(2) Day 0-4: multipotential stem cell posteriorly break up by former cell (Late primitive streak)
Culture medium (the similar culture medium of E8 or TeSR or other) is maintained to inhale when the convergence degree of hPSC reaches 40-60%
It removes, addition 500 μ L-1mL DPBS washing cell is primary, with 0.2-0.8mL/cm2Renal epithelial cell is added and breaks up complete medium
A is placed in 37 DEG C, 5%CO2Concentration, in the incubator of saturated humidity, continuous culture 4 days.Example of spatial compartmentalizationis, daily later
Replace culture medium.
Day 0-4 optimum experimental:
1. it includes the agent of Wnt signal activation and BMP Signal Regulation agent in complete medium A that renal epithelial cell, which breaks up,.The present invention
It is preferred that inhibiting glycogen synthase kinase-3beta (GSK-3 β) to activate Wnt signal dredging collateral.Inhibiting effective inhibitor of GSK-3 β has:
CHIR99021, SB216763, TWS119, LY2090314, Tideglusib, BIO-acetoxime etc..It is selected after present invention optimization
CHIR99021 is selected, concentration is 3-20 μM.The agent of BMP Signal Regulation includes BMP signal path activator and inhibitor in the present invention.
BMP activator is the substance, including BMP4, BMP2B and BMP7 etc. for activating BMP signal path.BMP inhibitor is to inhibit BMP letter
Chordin, Noggin, Follistatin, LDN193189, Dorsomorphin etc. may be selected in the substance of number access.The present invention
BMP4 is selected to activate BMP signal path, concentration 0-20ng/mL after optimization；LDN193189 is selected to inhibit BMP signal logical
Road, concentration 0-50nM.The induction point of renal epithelial cell is carried out using the renal epithelial cell differentiation complete medium A after optimization
Change, fine and close sheet of cell mass, cell mass tiling, and the smooth of the edge is presented in the 4th day cellular morphology.
(3) Day 4-6: rear portion original cell intermediate mesoderm (Posterior intermediate backward
Mesoderm) break up
After people's renal epithelial cell breaks up complete medium A culture 4 days, inhales and abandon culture medium, with 0.2-0.8mL/cm2People is added
Renal epithelial cell breaks up complete medium B, is placed in 37 DEG C, 5%CO2Concentration, in the incubator of saturated humidity, continuous culture 2 days,
Fresh differentiation complete medium B is replaced daily.
Day4-6 optimum experimental:
1. the main component that people's renal epithelial cell breaks up additive B in complete medium B are as follows: the activation of TGF-β signal path
Agent, common TGF-β signal path activator have TGF β -1, TGF β -2, TGF β -3, Activin A etc., select after present invention optimization
Select activator of the Activin A as TGF-β, concentration 5-50ng/mL.Processing cell 2 days, can be improved differentiation efficiency, control
System differentiation cost, simplifies experimental program.
(4) Day6-8: kidney mesenchymal cell (Metanephric mesenchyme) breaks up rear intermediate mesoderm backward
After people's renal epithelial cell breaks up complete medium B culture 2 days, inhales and abandon culture medium, according to 0.2-0.8mL/cm2It is added
People's renal epithelial cell breaks up complete medium C, is placed in 37 DEG C, 5%CO2Concentration in the incubator of saturated humidity, continuously cultivates 2
It, replaces fresh differentiation complete medium C daily.It can break up within 8th day to obtain rear kidney mesenchymal cell type, be filled between the rear kidney
Cell plastid is also known as kidney progenitor cells.The 7th day or the 8th day by cell dissociation after using kidney progenitor cells frozen stock solution by cell dissociation after freeze
It deposits with liquid nitrogen container, the cell after recovery can proceed with subsequent differentiation；Or 3D culture will be turned after the digestion of day8 kidney progenitor cells,
Obtain 3D kidney organoid.Specific experiment scheme is shown in " two, human pluripotent stem cells to renal epithelial cell directed differentiation -3D ".
Day6-8 optimum experimental:
1. cell dissociation buffer includes Trypsin (trypsase), Accutase (cell dissociation buffer), EDTA and other cells
Digestive pharmaceutical.Preferably, cell dissociation is carried out using 0.125-0.25%Trypsin, while isometric Trypsin need to be used
Inhibitor (trypsin inhibitor) terminates digestion.It is small to cell damage after vitellophag, can continue to cell cryopreservation or into
Row 3D culture.
2. the main component that people's renal epithelial cell breaks up addition of C in complete medium C are as follows: FGF signal path activator.
FGF signal path activator has FGF2, FGF8, FGF9, FGF10, FGF20 etc..Preferably, select FGF9 logical as FGF signal
The activator on road, concentration 5-50ng/mL.Processing cell 2 days, can be improved differentiation efficiency, and control differentiation cost simplifies experiment
Scheme.
(5) Day8-10: rear kidney mesenchymal cell breaks up to kidney duct shape aggregation precursor (Pre-tubular aggregate)
After people's renal epithelial cell breaks up complete medium C culture 2 days, inhales and abandon culture medium, according to 0.2-0.8mL/cm2It is added
People's renal epithelial cell breaks up complete medium D, is placed in 37 DEG C, 5%CO2Concentration in the incubator of saturated humidity, continuously cultivates 2
It, replaces fresh differentiation complete medium D daily.
Day8-10 optimum experimental:
1. the main component of additive D in culture medium D are as follows: FGF signal path activator and Wnt signal activation agent.FGF letter
Number Pathway Activation agent has FGF2, FGF8, FGF9, FGF10, FGF20 etc..Preferably, FGF9 is selected to activate FGF signal path,
Concentration is 5-25ng/mL.
Preparation method provided by the invention activates Wnt signal dredging collateral by inhibiting glycogen synthase kinase-3beta (GSK-3 β).
Inhibiting effective inhibitor of GSK-3 β has: CHIR99021, SB216763, TWS119, LY2090314, Tideglusib, BIO-
Acetoxime, NP031112, TWS119, CHIR-98014, AZD2858, AZD1080, SB415286 etc..After present invention optimization
CHIR99021 is selected to inhibit GSK-3 β, concentration 3-12uM.Differentiation efficiency, control differentiation can be improved in processing cell 2 days
Cost simplifies experimental program.
(6) Day10-13: kidney duct shape assembles precursor to kidney bubble (Renal vesicle) differentiation
After people's renal epithelial cell breaks up complete medium D culture 2 days, inhales and abandon culture medium, according to 0.2-0.8mL/cm2It is added
People's renal epithelial cell breaks up complete medium C, is placed in 37 DEG C, 5%CO2Concentration in the incubator of saturated humidity, continuously cultivates 3
It, replaces fresh differentiation complete medium C daily.
Day10-13 optimum experimental:
1. the main component that people's renal epithelial cell breaks up addition of C in complete medium C are as follows: FGF signal path activator.
FGF signal path activator has FGF2, FGF8, FGF9, FGF10, FGF20 etc..Preferably, select FGF9 logical as FGF signal
The activator on road, concentration 5-50ng/mL.Processing cell 2 days, can be improved differentiation efficiency, and control differentiation cost simplifies experiment
Scheme.
(7) Day13-20: kidney is steeped to be broken up to renal epithelial cell
It inhales and abandons culture medium, according to 0.2-0.8mL/cm2People's renal epithelial cell is added and breaks up basal medium E, is placed in 37 DEG C,
5%CO2Concentration in the incubator of saturated humidity, replaces fresh differentiation basal medium E daily.It can get maturation within about 5-7 days
Renal epithelial cell.Obtained renal epithelial cell has intuitive visible winding shape tubular structure, can directly carry out immunofluorescence
Dyeing detects the expression of each marker, can also carry out RNA extraction after cell cracking, each marker is carried out by way of qPCR
Detection.The subsequent test that can carry out renal drug toxicity and drug screening relevant to kidney trouble etc..
Day13-20 optimum experimental:
1. culture medium E that this programme uses by optimization, be added certain density Transferrin, insulin,
The compounds such as the albumen such as GlutaMAX additive and ethanol amine, Sodium Pyruvate, ammonium metavanadate, sodium selenite, manganese chloride, can
The mature differentiation for promoting renal epithelial cell, can maintain the culture of renal epithelial cell up to 3 months.
Two, human pluripotent stem cells are to renal epithelial cell directed differentiation -3D
The specific experiment scheme of Day1-8 is referring to " one, human pluripotent stem cells to renal epithelial cell directed differentiation -2D ".
Day8-10: the formation of embryoid body (EB) (kidney progenitor cells assemble precursor differentiation to kidney duct shape)
The kidney progenitor cells of 2D method culture to day8 are subjected to embryoid body and form experiment.Concrete operations are as follows: to day8 kidney ancestral
0.2-0.8mL/cm is added in cell2Cell dissociation buffer, after 37 DEG C of incubation 3-6min, softly blown and beaten with rifle, by cell suspension
It is transferred to 1.5mL centrifuge tube, brief centrifugation 5-10s inhales and abandons supernatant.0.2-0.8mL/cm is added2Rock containing a certain concentration inhibits
The differentiation complete medium D of agent is resuspended cell and is simultaneously transferred in the culture bottle of low absorbability, is placed in three-dimensional shaking table, and 37 DEG C, 5%
CO2Concentration, the interior culture of the incubator of saturated humidity.Cell density can be 0.1-5x106/mL；The revolving speed of shaking table can be 10-
20rpm；The time of culture can be 8-32 hours.
Day8-10 optimum experimental:
1.Rock inhibitor includes Y-27632, Blebbistatin, HA100 etc..Preferably, Rock inhibitor is Y-
27632, concentration 10uM.
2. cell dissociation buffer includes Trypsin, Accutase, EDTA or other cell dissociation buffers.Preferably, it uses
0.125-0.25%Trypsin carries out cell dissociation, while isometric Trypsin inhibitor need to be used to terminate digestion, disappears
It is small to cell damage after change cell, it can be cultivated for 3D and the cell of higher vigor be provided；
3. low absorbability 3D culture bottle includes T25 Flask, T75 Flask, T225 Flask and other low absorbabilities
3D Tissue Culture Flask.Or coating prevents cell adherent for the low absorbability matrix of 3D culture.Preferably, using Poly-
Hema (more poly 2-hydroxyethyl methacrylate rouge) carries out coating use to culture bottle, and effect is more preferable, and EB can keep suspending and not paste
Wall.
Day10-13: kidney duct shape assembles precursor to kidney bubble (Renal vesicle) differentiation
After people's renal epithelial cell breaks up complete medium D culture 2 days, inhales and abandon culture medium, according to 0.2-0.8mL/cm2It is added
People's renal epithelial cell breaks up complete medium C, is placed in 37 DEG C, 5%CO2Concentration in the incubator of saturated humidity, continuously cultivates 3
It.
Day10-13 optimum experimental:
1. the main component that people's renal epithelial cell breaks up addition of C in complete medium C are as follows: FGF signal path activator.
FGF signal path activator has FGF2, FGF8, FGF9, FGF10, FGF20 etc..Preferably, select FGF9 logical as FGF signal
The activator on road, concentration 5-50ng/mL.Processing cell 2 days, can be improved differentiation efficiency, and control differentiation cost simplifies experiment
Scheme.
Day13-20: kidney is steeped to be broken up to renal epithelial cell
After people's renal epithelial cell breaks up complete medium C culture 3 days, inhales and abandon culture medium, according to 0.2-0.8mL/cm2It is added
People's renal epithelial cell breaks up basal medium E, is placed in 37 DEG C, 5%CO2Concentration about 2-4 days can in the incubator of saturated humidity
To obtain the apparent 3D kidney organoid of renal epithelial cell structure.Obtained 3D kidney organoid has intuitive visible winding shape
Tubulose renal epithelial cell structure can carry out the expression that immunofluorescence dyeing detects each marker, can also carry out RNA after cell cracking
It extracts, the detection of each marker is carried out by way of qPCR, it is subsequent to carry out kidney diaseases model foundation, renal drug toxicity
Test and drug screening relevant to kidney trouble etc..
Day13-20 optimum experimental:
1. people's renal epithelial cell differentiation basal medium E that this programme uses is added certain density by optimization
The albumen such as Transferrin, insulin, GlutaMAX additive and ethanol amine, Sodium Pyruvate, ammonium metavanadate, selenous acid
The compounds such as sodium, manganese chloride, can promote the mature differentiation of renal epithelial cell, and maintain renal epithelial cell stable structure.
The ingredient of above-mentioned people's renal epithelial cell differentiation basal medium E is as shown in table 2.
Table 2
Other medium components are shown in Table 3 and table 4 in the present invention.
Table 3
Table 4
In a specific embodiment of the invention, above-mentioned human pluripotent stem cells pass through disclosed in patent CN 108085299A
It is prepared by method.
It should be appreciated that those skilled in the art can also select arbitrary human pluripotent stem cells business cell line as needed
Or cell strain completes the present invention, and within the scope of the present invention.
The renal epithelial cell that the above method is prepared is detected, is chosen any one kind of them in following methods:
Method one: use RT-qPCR technology: proves the obtained cell of differentiation for CDH2+/CDH1+/PODXL+/kidney on
Chrotoplast.Wherein, CDH2 is the significant index of proximal tubule, and CDH1 is the significant index of Heng Lishi ring and distal tubule,
PODXL is the significant index of glomerulus progenitor cells.
Preferably, kit used in extract RNA is RNAprep Pure Cell/Bacteria Kit, and producer is
TIANGEN, article No. are DP430；Reverse transcription used kit is Reverse Transcriptase, producer Vazyme, article No.
It is R223-01；RT-QPCR used kit is TransStart Top Green qPCR SuperMix, and producer is
Transgen, article No. are AQ131.
Wherein, the primer sequence of RT-qPCR is as follows:
CDH1-F:ATTTTTCCCTCGACACCCGAT (SEQ ID NO:1)；
CDH1-R:TCCCAGGCGTAGACCAAGA (SEQ ID NO:2)；
CDH2-F:TGCGGTACAGTGTAACTGGG (SEQ ID NO:3)；
CDH2-R:GAAACCGGGCTATCTGCTCG (SEQ ID NO:4)；；
PODXL-F:AGCTAAACCTAACACCACAAGC (SEQ ID NO:5)；
PODXL-R:TGAGGGGTCGTCAGATGTTCT (SEQ ID NO:6).
Method two: the kidney epithelium that the cell that differentiation obtains is LTL+/CDH1+/PODXL+ is proved using immunofluorescence dyeing
Cell.Wherein, LTL is the significant index of proximal tubule, and CDH1 is the significant index of Heng Lishi ring and distal tubule,
PODXL is the significant index of glomerulus progenitor cells, and DAPI is the dyeing of nucleus.
Preferably, the antibody information of immunofluorescence: Lotus tetragonolobus lectin (LTL),
Vectorlabs, #FL-1321；E-cadherin (CDH1), Abcam, #ab40772；PODXL, R&D Systems, #
AF1658；Donkey anti-Goat IgG, cy3, #705-165-147；Donkey anti-Goat IgG, FITC, #705-
095-147；Donkey anti-Rabbit IgG, cy3, #711-165-152.
Further, inventor has found that the above method is prepared renal epithelial cell and can generate renal toxicity response to cis-platinum.
Wherein, kim1 is the significant index of proximal tubule damage, and LTL is the significant index of proximal tubule, and DAPI is nucleus
Dyeing.
In a specific embodiment of the invention, antibody information is as follows: kim1, R&D Systerm, #AF1750；Lotus
Tetragonolobus lectin (LTL), Vectorlabs, #FL-1321；Donkey anti-Goat IgG, cy3, #705-
165-147。
Further, inventor has found that renal epithelial cell is prepared through forskolin (adenyl cyclase in the above method
Activator) vesica can be generated after induction, and the increase of vesica is able to suppress using Rapamycin (rapamycin).At this
In one specific embodiment of invention, which includes: Forskolin, MedChen Express, #HY15371；
Rpamycin, SIGMA, #V900930.
Third aspect of the present invention discloses a kind of renal epithelial cell, is prepared by above-mentioned method.
The 4th aspect of the present invention discloses a kind of cell mass, is enriched with above-mentioned renal epithelial cell.
The 5th aspect of the present invention discloses a kind of drug for treating kidney trouble, and the drug includes above-mentioned kidney epithelium
Cell.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination, and without departing from structure of the invention
Think of and protection scope.
Key technology of the invention includes the following:
The present invention has following remarkable advantage and effect compared with the existing technology:
1) the people's renal epithelial cell differentiation basal medium E definite ingredients provided in preparation method of the present invention, active principle
Stabilization is not degradable, and differentiation efficiency is high.
2) agent formulations used in atomization are clear, using the more efficient economy of small molecule compound, make the knot of differentiation
Fruit is more stable；While guaranteeing differentiation efficiency, the dosage of preparation method growth factor of the present invention is low, dramatically saves scientific research
Cost.
3) it in renal epithelial cell preparation method provided by the invention, can be carried out in a manner of 2D and 3D.
4) kidney progenitor cells frozen stock solution provided by the invention, Cell viability is high after freezing the recovery of kidney progenitor cells, and still retains phase
The expression for closing mark, can continue to be divided into renal epithelial cell.
The present invention has following remarkable advantage and effect compared with the existing technology:
First, preparation method provided by the invention optimizes by test of many times, can break up within the shorter time and be had
There is the kidney organoid of mature renal epithelial cell structure.It shortens on time nearly one week, greatly saves compared with the existing technology
Time cost.
Second, the nephrons progenitor cells efficiency that the preparation method provided through the invention is broken up reaches 90% or more (ginseng
According to attached drawing 5), substantially increase the yield of renal epithelial cell；It can satisfy the test of kidney diaseases model foundation, renal drug toxicity
And the scientific experiments demand such as drug screening relevant to kidney trouble.
Third, the 4 kinds of additives provided in preparation method of the present invention are by repeatedly screening, it is ensured that definite ingredients, and can
Realize more efficient, inexpensive differentiation, efficiently solve the prior art differentiation it is at high cost, it is complicated for operation the problems such as.
4th, preparation method provided by the invention has been realized in the differentiation on a variety of hPSC cell strains, has wider
General adaptability.
5th, it is thin can effectively to freeze the kidney ancestral that differentiation the obtains for 7-8 days for kidney progenitor cells frozen stock solution provided by the invention
Born of the same parents, and motility rate is high after recovery, and kidney progenitor cell marker index is still expressed, and is had and is continued to be divided into the latent of renal epithelial cell
Energy.
Detailed description of the invention
Fig. 1 shows the flow chart of preparation method one embodiment of the present invention.
Fig. 2 shows the 0th day (hPSC) of 2D differentiation culture, the 4th day, the 6th day, the 8th day, the 13rd day, the 20th day specific
Cellular morphology.
Fig. 3 shows the cellular morphology of the 13rd day of 3D differentiation culture, the 15th day.
Fig. 4 is that different cell strains (including: EGFP-ipsc, the ipsc of polycystic kindey patient, the ipsc of Healthy People) differentiation obtains
Renal epithelial cell.
Fig. 5 is the cell progress SIX+ immunofluorescent staining obtained for 7-8 days in the embodiment of the present invention to differentiation the, with
And flow cytomery SIX2+ expression efficiency.
Fig. 6 show with different frozen stock solutions to kidney progenitor cells freeze processing after, then after recovering kidney progenitor cells cell
Survival rate test
Fig. 7 show with different frozen stock solutions to kidney progenitor cells freeze processing after, then after recovering kidney progenitor cells SIX2 flows
Formula cell instrument testing result figure.
Fig. 8 is shown using RT-qPCR technology to differentiation the 19th day (D19) and the 21st day (D21) resulting renal epithelial cell
It is detected, shows that (NC is negative control group, the NC that CDH1 and PODXL are used in figure for the expression of CDH2+/CDH1+/PODXL+
It is epidermal cell for MSC, the CDH2 NC used).
Fig. 9, which is shown, detects the 20th day resulting renal epithelial cell of differentiation using immunofluorescence dyeing technology, shows
The expression of LTL+/CDH1+/PODXL+ (BF is indicated and shot off field in figure).
Figure 10 is drug toxicity response result figure of the renal epithelial cell obtained in the embodiment of the present invention to cis-platinum.
Figure 11 is the vesica that renal epithelial cell obtained in the embodiment of the present invention generates, and vesica can be by rapamycin
(Rapamycin) inhibit figure.
Specific embodiment
Technical solution of the present invention is described in detail with reference to the accompanying drawings and examples, but therefore will be not of the invention
It is limited among the embodiment described range.
In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or says according to commodity
Bright book selection.The reagents and materials used in the present invention are commercially available.
Embodiment 1
Present embodiment discloses a kind of methods for preparing renal epithelial cell, comprising the following steps:
S1: multipotential stem cell posteriorly former cell differentiation；
S2: intermediate mesoderm breaks up rear portion original cell backward；
S3: rear intermediate mesoderm breaks up to kidney progenitor cells；
S4: kidney progenitor cells assemble precursor differentiation to kidney duct shape；
S5: kidney duct shape assembles precursor and steeps differentiation to kidney；
S6: kidney is steeped to be broken up to renal epithelial cell.
Specifically, flow chart is as shown in Figure 1, steps are as follows:
(1) culture (D-3-D0) of multipotential stem cell
Multipotential stem cell used in experiment (expresses various versatility markers, and can by stringent versatility verifying
Formed in immune-deficient mice body comprising it is interior, in, the teratoma of outer three germinal layers).Multipotential stem cell maintains culture at it
It is normally cultivated in base, the similar culture medium of culture medium E8 or TeSR or other used.Human pluripotent stem cells pass through in the embodiment
It is prepared by method disclosed in patent CN 108085299A.
It is cultivated after optimized using Nova culture medium, can stablize and maintain the long-term cultivation of hPSC in vitro.
Rock inhibitor is added in above-mentioned maintenance culture medium.Rock inhibitor is Y-27632, and concentration is 10 μM；
The inoculum density of hPSC can be 1-10 × 104cells/cm2。
The present embodiment carries out cell dissociation using Accutase.Cell facilitates counting in unicellular after vitellophag,
And it is small to cell damage.
(2) multipotential stem cell posteriorly former cell differentiation (Day 0-4)
Culture medium (the similar culture medium of E8 or TeSR or other) is maintained to inhale when the convergence degree of hPSC reaches 40-60%
It removes, addition 500 μ L-1mL DPBS washing cell is primary, with 0.2-0.8mL/cm2Renal epithelial cell is added and breaks up complete medium
A is placed in 37 DEG C, 5%CO2Concentration, in the incubator of saturated humidity, continuous culture 4 days.Example of spatial compartmentalizationis, daily later
Replace culture medium.
It includes the agent of Wnt signal activation and BMP Signal Regulation agent in complete medium A that renal epithelial cell, which breaks up,.The present embodiment
Wnt signal dredging collateral is activated using glycogen synthase kinase-3beta (GSK-3 β) is inhibited.The present invention selects CHIR99021 as inhibition
The inhibitor of GSK-3 β, and concentration is 3-20 μM.The agent of BMP Signal Regulation includes BMP signal path activator and suppression in the present invention
Preparation.BMP activator is the substance for activating BMP signal path, and BMP inhibitor is the substance for inhibiting BMP signal path.This implementation
Example selects BMP4 to activate BMP signal path, concentration 0-20ng/mL；LDN193189 is selected to inhibit BMP signal path, it is dense
Degree is 0-50nM.
Broken up using the induction that the renal epithelial cell differentiation complete medium A after optimization carries out renal epithelial cell, the 4th day
Fine and close sheet of cell mass, cell mass tiling, and the smooth of the edge is presented in cellular morphology.
(3) the former cell in rear portion intermediate mesoderm differentiation (Day 4-6) backward
After people's renal epithelial cell breaks up complete medium A culture 4 days, inhales and abandon culture medium, with 0.2-0.8mL/cm2People is added
Renal epithelial cell breaks up complete medium B, is placed in 37 DEG C, 5%CO2Concentration, in the incubator of saturated humidity, continuous culture 2 days,
Fresh people's renal epithelial cell differentiation complete medium B is replaced daily.
It includes additive B, the ingredient of additive B in complete medium B that people's renal epithelial cell, which breaks up, are as follows: TGF-β signal is logical
Road activator.The present embodiment selects Activin A as the activator of TGF-β, concentration 5-50ng/mL.Processing cell 2 days,
Differentiation efficiency can be improved, control differentiation cost simplifies experimental program.
(4) afterwards intermediate mesoderm to kidney progenitor cells break up (Day6-8)
After people's renal epithelial cell breaks up complete medium B culture 2 days, inhales and abandon culture medium, according to 0.2-0.8mL/cm2It is added
People's renal epithelial cell breaks up complete medium C, is placed in 37 DEG C, 5%CO2Concentration in the incubator of saturated humidity, continuously cultivates 2
It, replaces fresh differentiation complete medium C daily.It can break up to obtain kidney progenitor cells within 8th day.
The 7th day or the 8th day by cell dissociation after using kidney progenitor cells frozen stock solution by cell dissociation after freeze and liquid nitrogen container
In, the cell after recovery can proceed with subsequent differentiation.
Cell dissociation is carried out using 0.125-0.25%Trypsin, while isometric Trypsin inhibitor need to be used
Terminate digestion.It is small to cell damage after vitellophag, it can continue to cell cryopreservation.
People's renal epithelial cell breaks up the ingredient of addition of C in complete medium C are as follows: FGF signal path activator.This implementation
Example selects FGF9 as the activator of FGF signal path, concentration 5-50ng/mL.Differentiation effect can be improved in processing cell 2 days
Rate, control differentiation cost, simplifies experimental program.
(5) afterwards kidney mesenchymal cell to kidney duct shape assemble precursor differentiation (Day8-10)
After people's renal epithelial cell breaks up complete medium C culture 2 days, inhales and abandon culture medium, according to 0.2-0.8mL/cm2It is added
People's renal epithelial cell breaks up complete medium D, is placed in 37 DEG C, 5%CO2Concentration in the incubator of saturated humidity, continuously cultivates 2
It, replaces fresh differentiation complete medium D daily.
People's renal epithelial cell breaks up the ingredient of additive D in complete medium D are as follows: FGF signal path activator and Wnt letter
Number activator.The present embodiment selects FGF9 to activate FGF signal path, concentration 5-25ng/mL.The present embodiment is by inhibiting sugar
Former 3 β of synthase kinase (GSK-3 β) activates Wnt signal dredging collateral.CHIR99021 is selected to inhibit GSK-3 β, concentration 3-12uM.
Processing cell 2 days, can be improved differentiation efficiency, and control differentiation cost simplifies experimental program.
(6) kidney duct shape assembles precursor to kidney bubble differentiation (Day10-13)
After people's renal epithelial cell breaks up complete medium D culture 2 days, inhales and abandon culture medium, according to 0.2-0.8mL/cm2It is added
People's renal epithelial cell breaks up complete medium C, is placed in 37 DEG C, 5%CO2Concentration in the incubator of saturated humidity, continuously cultivates 3
It, replaces fresh differentiation complete medium C daily.
People's renal epithelial cell breaks up the ingredient of addition of C in complete medium C are as follows: FGF signal path activator.Selection
Activator of the FGF9 as FGF signal path, concentration 5-50ng/mL.Processing cell 2 days, can be improved differentiation efficiency, control
Break up cost, simplifies experimental program.
(7) kidney, which is steeped to renal epithelial cell, breaks up (Day13-20)
It inhales and abandons culture medium, according to 0.2-0.8mL/cm2People's renal epithelial cell is added and breaks up basal medium E, is placed in 37 DEG C,
5%CO2Concentration in the incubator of saturated humidity, replaces fresh differentiation basal medium E daily.It can get maturation within about 5-7 days
Renal epithelial cell.Obtained renal epithelial cell has intuitive visible winding shape tubular structure, can directly carry out immunofluorescence
Dyeing detects the expression of each marker, can also carry out RNA extraction after cell cracking, each marker is carried out by way of qPCR
Detection.The subsequent test that can carry out renal drug toxicity and drug screening relevant to kidney trouble etc..
People's renal epithelial cell differentiation basal medium E in the present embodiment is added certain density by optimization
The albumen such as Transferrin, insulin, GlutaMAX additive and ethanol amine, Sodium Pyruvate, ammonium metavanadate, selenous acid
The compounds such as sodium, manganese chloride can promote the mature differentiation of renal epithelial cell, the culture of renal epithelial cell can be maintained up to 3
A month.
The component of above-mentioned kidney progenitor cells frozen stock solution is as shown in table 1 below.
Table 1
The ingredient of above-mentioned people's renal epithelial cell differentiation basis E is as shown in table 2.
Table 2
Other medium components are shown in Table 3 and table 4 in the present embodiment.
Table 3
Table 4
Wherein break up the 0th day (hPSC) of culture, the 4th day, the 6th day, the 8th day, the 13rd day, the 20th day specific cell shape
State is shown in attached drawing 2.Present embodiments provide the system that one kind can quickly, efficiently, easily be renal epithelial cell from hPSC directed differentiation
Preparation Method, medium component is clear, and divergaence time is short.
Embodiment 2
Present embodiment discloses a kind of method for preparing renal epithelial cell, it is unique from embodiment 1 unlike: step (5)
" rear kidney mesenchymal cell assembles precursor differentiation (Day8-10) to kidney duct shape " uses the culture of 3D method.The following steps are included:
Obtained kidney progenitor cells are subjected to embryoid body (EB) and form experiment, concrete operations are as follows: the kidney ancestral obtained to differentiation is thin
0.2-0.8mL/cm is added in born of the same parents2Cell dissociation buffer, after 37 DEG C of incubation 3-6min, softly blown and beaten with rifle, cell suspension turned
1.5mL centrifuge tube is moved to, brief centrifugation 5-10s inhales and abandons supernatant.0.2-0.8mL/cm is added2The inhibitor of Rock containing a certain concentration
Differentiation complete medium D be resuspended and cell and be transferred in the culture bottle of low absorbability, be placed in three-dimensional shaking table, 37 DEG C, 5%CO2
Concentration, the interior culture of the incubator of saturated humidity.Cell density can be (0.1-5) x106/mL；The revolving speed of shaking table can be 10-
20rpm；The time of culture can be 8-32 hours.
Rock inhibitor is Y-27632, concentration 10uM.
Cell dissociation is carried out using 0.125-0.25%Trypsin, while isometric Trypsin inhibitor need to be used
It terminates and digests, it is small to cell damage after vitellophag, it can be cultivated for 3D and the cell of higher vigor be provided；
Low absorbability 3D culture bottle includes T25 Flask, T75 Flask, T225 Flask and other low absorbabilities 3D
Tissue Culture Flask.Or coating prevents cell adherent for the low absorbability matrix of 3D culture.Using Poly-Hema to culture
Bottle carries out coating use, and effect is more preferable, and EB can keep suspending not adherent.3D breaks up the cell shape of the 13rd day of culture, the 15th day
State is as shown in Figure 3.
Embodiment 3
Present embodiment discloses the method for preparing renal epithelial cell, it is unique from embodiment 1 unlike: multipotential stem cell
Source is different, the iPSC of respectively EGFP-iPSC, the iPSC of polycystic kindey patient and Healthy People.Three kinds of cell strains are getable
Typical renal epithelial cell structure (see Fig. 3) illustrates that differentiation method disclosed by the invention has the universality to a variety of hPSC.
Embodiment 4
The present embodiment carries out SIX2 immunofluorescence dye for the cell that the obtains for 7-8 days is broken up in method disclosed in embodiment 1
Color and flow cytomery, are as a result shown in Fig. 4, wherein Immunofluorescent Antibody information is SIX2, Proteintech, 11562-1-
AP。
Embodiment 5
The present embodiment studies the shadow of kidney progenitor cells activity and differentiation capability that different frozen stock solutions obtains embodiment 1
It rings, specifically, using kidney progenitor cells frozen stock solution disclosed in embodiment 1 and commercially available three kinds of frozen stock solutions to carry out respectively kidney progenitor cells
Liquid nitrogen cryopreservation.It recovers kidney progenitor cells after a week are frozen with four kinds of frozen stock solutions respectively, calculates Cell viability, as a result as schemed
Shown in 6, from fig. 6, it can be seen that using cell after the kidney progenitor cells recovery that kidney progenitor cells frozen stock solution freezes disclosed in embodiment 1
Motility rate highest, wherein Cell viability=viable count/total cell number.
It is cells frozen storing liquid disclosed in 201610135615.4 that above-mentioned frozen stock solution 1, which is number of patent application,；Frozen stock solution 2 is patent
Application No. is cells frozen storing liquids disclosed in 201710932562.3；Frozen stock solution 3 is that number of patent application is 201610068837.9 public affairs
The cells frozen storing liquid opened.
Then to after recovery kidney progenitor cells carry out SIX2 flow cytomery, as a result see Fig. 7, from Fig. 7 it can be found that
It is more using the cell for expressing SIX2 after the kidney progenitor cells recovery that kidney progenitor cells frozen stock solution freezes disclosed in embodiment 1.
Illustrate kidney progenitor cells can be made effectively to be saved using the frozen stock solution in embodiment 1, Cell viability after recovery
Expression that is high and still retaining correlating markings, can continue to be divided into renal epithelial cell.
Embodiment 5
The present embodiment is by RT-qPCR technology to breaking up the 19th day (D19) in embodiment 1 and the 21st day (D21) resulting
Cell is detected, as a result as shown in Figure 8.Wherein, CDH2 is the significant index of proximal tubule, and CDH1 is for Heng Lishi ring and far
The significant index of tubule is held, PODXL is the significant index of glomerulus progenitor cells.Kit used in extract RNA is
RNAprep Pure Cell/Bacteria Kit, producer TIANGEN, article No. is DP430；Reverse transcription used kit is
Reverse Transcriptase, producer Vazyme, article No. are R223-01；RT-QPCR used kit is
TransStart Top Green qPCR SuperMix, producer Transgen, article No. is AQ131.
Primer sequence in RT-qPCR is as follows:
CDH1-F:ATTTTTCCCTCGACACCCGAT (SEQ ID NO:1)
CDH1-R:TCCCAGGCGTAGACCAAGA (SEQ ID NO:2)
CDH2-F:TGCGGTACAGTGTAACTGGG (SEQ ID NO:3)
CDH2-R:GAAACCGGGCTATCTGCTCG (SEQ ID NO:4)
PODXL-F:AGCTAAACCTAACACCACAAGC (SEQ ID NO:5)
PODXL-R:TGAGGGGTCGTCAGATGTTCT (SEQ ID NO:6).
Embodiment 6
The present embodiment is detected using immunofluorescence dyeing technology to breaking up the 20th day obtained cell in embodiment 1,
Prove that obtained cell is the renal epithelial cell of LTL+/CDH1+/PODXL+.Specific data are shown in attached drawing 9.Wherein, LTL is proximal end
The significant index of tubule, CDH1 are the significant index of Heng Lishi ring and distal tubule, and PODXL is the mark of glomerulus progenitor cells
Will index, DAPI are the dyeing of nucleus.The antibody information of immunofluorescence: Lotus tetragonolobus lectin
(LTL), Vectorlabs, #FL-1321；E-cadherin (CDH1), Abcam, #ab40772；PODXL, R&D Systems, #
AF1658；Donkey anti-Goat IgG, cy3, #705-165-147；Donkey anti-Goat IgG, FITC, #705-
095-147；Donkey anti-Rabbit IgG, cy3, #711-165-152；
Embodiment 7
The present embodiment carries out drug toxicity test to renal epithelial cell obtained in embodiment 1, and discovery embodiment 1 obtains
Renal epithelial cell structure is mature, and wherein proximal tubule has the drug toxicity responsing reaction to cis-platinum.
The cisplatin medicine information used: Cisplatin, SIGMA, #P4394-25MG.Kidney is cultivated using basal medium E
Epithelial cell for 24 hours after, fixed cell carries out immunofluorescence dyeing, and experimental result is shown in Figure 10.Wherein, the final concentration of 10- of cis-platinum
50uM, Kim1 are the significant indexs of proximal tubule damage, and LTL is the significant index of proximal tubule, and DAPI is nucleus
Dyeing.Immunofluorescent Antibody information: Lotus tetragonolobus lectin (LTL), Vectorlabs, #FL-1321；
Kim 1, R&D system, #AF1750；Donkey anti-Goat IgG, cy3, #705-165-147.
This example demonstrates that the renal epithelial cell that embodiment 1 obtains can be used in the test of renal drug toxicity.
Embodiment 8
The present embodiment carries out the relevant pharmaceutically-active research of kidney trouble to renal epithelial cell obtained in embodiment 1.
Experiment is divided into control group (control) and medicine group, and control group is handled renal epithelial cell using physiological saline, medicine group
Renal epithelial cell is handled using the Rapamycin of equivalent.The result is shown in Figure 11, experiment discovery Rapamycin have vesica
Inhibiting effect.The renal epithelial cell that embodiment 1 is prepared can be used for the scientific experiments such as drug screening relevant to kidney trouble
It needs.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
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Claims (15)
1. a kind of renal epithelial cell expresses CDH1+ and PODXL+, one of CDH2+ and LTL+ are also at least expressed.
2. a kind of method for preparing renal epithelial cell, which comprises the following steps:
S1: multipotential stem cell posteriorly former cell differentiation；
S2: intermediate mesoderm breaks up rear portion original cell backward；
S3: rear intermediate mesoderm breaks up to kidney progenitor cells；
S4: kidney progenitor cells assemble precursor differentiation to kidney duct shape；
S5: kidney duct shape assembles precursor and steeps differentiation to kidney；
S6: kidney is steeped to be broken up to renal epithelial cell.
3. according to the method described in claim 2, it is characterized in that, the S1 includes:
S11: the supernatant of human pluripotent stem cells is absorbed, and renal epithelial cell differentiation complete medium A induction differentiation is added, described
Renal epithelial cell differentiation complete medium A includes that Wnt signal path activator, BMP4 signal pathway inhibitor and BMP4 signal are logical
Road activator；
S12: replacing culture medium daily, cultivates 4-5 days.
4. according to the method described in claim 2, it is characterized in that, the S2 includes:
S21: absorbing the supernatant of the former cell in rear portion, and renal epithelial cell differentiation complete medium B induction differentiation, the kidney is added
It includes TGF-β signal path activator that epithelial cell, which breaks up complete medium B,；
S22: replacing culture medium daily, cultivates 2-3 days.
5. according to the method described in claim 2, it is characterized in that, the S3 includes:
S31: renal epithelial cell differentiation complete medium C induction differentiation, the kidney is added in the supernatant of intermediate mesoderm after absorption
It includes FGF signal path activator that epithelial cell, which breaks up complete medium C,；
S32: replacing culture medium daily, cultivates 2-3 days.
6. according to the method described in claim 5, it is characterized in that, further including step S33: by the obtained kidney progenitor cells of differentiation into
Row freezes.
7. according to the method described in claim 6, it is characterized in that, being frozen using frozen stock solution to kidney progenitor cells in S33, institute
Stating frozen stock solution includes the DMSO and recombinant human serum albumin that percentage by volume is respectively 5-20% and 0-10%.
8. according to the method described in claim 2, it is characterized in that, S4 culture by the way of 2D, comprising the following steps:
S41: absorbing the supernatant of kidney progenitor cells, and renal epithelial cell differentiation complete medium D induction differentiation, the kidney epithelium is added
Cell differentiation complete medium D includes FGF signal path activator and Wnt signal path activator；
S42: replacing culture medium daily, cultivates 2-3 days.
9. according to the method described in claim 2, it is characterized in that, S4 culture by the way of 3D, comprising the following steps:
Kidney progenitor cells are digested, is resuspended with the renal epithelial cell differentiation complete medium D containing Rock inhibitor, is placed in three-dimensional and shakes
Suspend culture on bed.
10. according to the method described in claim 2, it is characterized in that, the S5 includes:
S51: absorbing the supernatant of kidney duct shape aggregation precursor, and renal epithelial cell differentiation complete medium C induction differentiation is added, described
It includes FGF signal path activator that renal epithelial cell, which breaks up complete medium C,；
S52: replacing culture medium daily, cultivates 2-3 days.
11. according to the method described in claim 2, it is characterized in that, the S6 includes:
S61: absorbing the supernatant of kidney bubble, and renal epithelial cell differentiation basal medium E induction differentiation is added；
S62: replacing culture medium daily, cultivates 5-7 days.
12. according to the method for claim 11, which is characterized in that the renal epithelial cell breaks up basal medium E and includes
Glutamine, human transferrin and insulin.
13. a kind of renal epithelial cell, the method as described in any one of claim 2-12 is prepared.
14. a kind of cell mass, which is characterized in that be enriched with renal epithelial cell described in claim 1 or 13.
15. a kind of drug for treating kidney trouble, which is characterized in that the drug includes kidney described in claim 1 or 13
Epithelial cell.
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